Multimodular Analysis of Angiogenic Biomarkers in the Primary Treatment of Multiple Myeloma: Correlative Studies of the BiRD Trial.

Biomarkers that predict the angiogenic propensity of multiple myeloma and the impact of therapy on angiogenesis remain unknown. Nevertheless, many putative anti-angiogenic agents are currently being evaluated in clinical trials. We describe the results of a multimodular analysis of angiogenic biomarkers including a novel in vitro functional assay, the human umbilical vein endothelial cell (HUVEC)-based angiogenic scale (HBAS), to assess the overall angiogenic activity in the plasma of patients with multiple myeloma. Plasma fractionated from the peripheral blood of myeloma patients was added to primary HUVEC and cultured under serum-free conditions for 24 hours. Angiogenic morphology was then examined and scored using a six-point angiogenic scale. Analysis of a cohort of ten consecutive patients with multiple myeloma undergoing the BiRD protocol (Biaxin, Revlimid and dexamethasone) showed that the majority had higher HBAS scores at baseline than age-matched normal subjects. Plasma samples from stage IIIA disease consistently scored higher than those from stage II disease on the functional angiogenic scale (mean score of 2.8 versus 1.0, p<0.005), suggesting a more pro-angiogenic state for further advanced disease. The HBAS also correlated with increased levels of plasma and platelet vascular endothelial growth factor-A (VEGF-A) as quantified by ELISA. Interestingly, VEGF-A levels in platelet lysate were significantly higher than those in plasma and more closely paralleled the functional HBAS scores. In addition, two-color flow cytometric analysis of cellular biomarkers demonstrated an angiogenic switch defined by increased mobilization of circulating CD133⁺VEGFR2⁺ endothelial progenitor cells in patients with active disease as compared to normal subjects (2.2-fold increase; p<0.005). Treatment with BiRD effectively attenuated all tested angiogenic parameters and correlated with a positive clinical response. Collective assessment of angiogenic biomarkers may be a promising resource for predicting clinical outcomes and for validating the potential impact of anti-angiogenic therapy in multiple myeloma.